Petroleum serves not only as the primary source of fuel but also as the raw material for production of various polymers used by the plastics industry. The finite nature of the petroleum reserves and the negative environmental impact from the use of petroleum has shifted attention towards alternate, renewable source of fuels and chemicals as replacements for petroleum (23). Fermentation of carbohydrates has been shown to produce various short chain hydroxy acids as well as other chemicals that can be polymerized to produce plastics with differing physical and chemical properties. Among these fermentation products, lactic acid stands out as a primary chemical that can be a starting material for manufacture of biodegradable, renewable plastics with minimal environmental impact; CO2 neutral. Fermentation of sugars to lactic acid dates back to pre-historic times and commercial production of lactic acid using pure bacterial cultures started as early as 1895 (3). Although lactic acid is primarily used by food and pharmaceutical industries, lactic acid derived biopolymer production is expected to outstrip these uses provided the cost of production of lactic acid based polymer is comparable to the polymers derived from petrochemicals (8, 14, 18).
Lactic acid is condensed into lactide, purified and polymerized to polylactide (PLA), a thermoplastic (18, 22). By judicial mixing of the D(−)- and L(+)-lactic acid, polymers with various physical and thermochemical properties can be produced. Although lactic acid can be synthesized from petroleum, the product is a mixture of the two isomers and is not suitable for PLA production. Optically pure lactic acid required for PLA production is produced only by microbial fermentation (14). Various lactic acid bacteria, Lactobacillus, Lactococcus, etc., produce L(+)-lactic acid at high yield and titer from fermentable sugars such as glucose and sucrose (6, 14). Their nutritional requirements are complex and their growth temperature range between 30° C. and 35° C. Microorganisms that produce D(−)-lactic acid as the primary fermentation product has been described and is currently being used by industry (25, 34, 36) (12). Lactic acid fermentation by these microbial biocatalysts is currently conducted at 30-37° C. and raising the growth and fermentation temperature to 50-55° C. is expected to minimize contamination of large scale industrial fermentations (1). In order to reduce the cost of lactic acid production and also to eliminate the use of food carbohydrates as feedstock for lactic acid production, alternate sources of fermentable sugars and microbial biocatalysts are being developed. Lignocellulosic biomass is an attractive source of sugars; glucose, xylose, etc. However, the lactic acid bacteria used by the industry lacks the ability to ferment pentoses efficiently to lactic acid although there are several attempts to improve the xylose fermentation property of these lactic acid bacteria (25, 31).
Bacillus coagulans is a sporogenic lactic acid bacterium that grows at 50-55° C. and pH 5.0 and ferments both hexoses and pentoses (10, 27). This bacterium has been shown to produce L(+)-lactic acid at concentrations as high as 180 g/L in fed-batch fermentations from both glucose and xylose and is also an excellent candidate for simultaneous saccharification and fermentation of cellulose to optically pure lactic acid (26). B. coagulans is, generally, recalcitrant to genetic engineering and methods for producing pure lactic acid are needed (particularly using genetically engineered microorganisms that do not contain exogenous nucleic acid sequences). One aspect of the invention disclosed herein provides a general method for engineering this genetically recalcitrant bacterium. Using the disclosed method and growth-based selection, the fermentation product of B. coagulans strain P4-102B is changed from L(+)-lactic acid to D(−)-lactic acid. The engineered biocatalyst produced about 90 g/L of D(−)-lactic acid in less than 48 hours at 50° C.